High throughput systems have been used to generate affinity tagged recombinant proteins to assist in their immobilization and purification, to study their function and structure. Although a number of tags have been developed, those that can be used efficiently for both purification and immobilization are limited; and biotinylation offers potential for both tasks. The tight binding of biotin to avidin and streptavidin results in capturing of biotinylated biomolecules. The availability of a form of streptavidin in appropriate quantities that binds reversibly, such that it can be used for capture and/or purification of biotinylated proteins in acceptable yield would significantly extend potential applications of biotin-streptavidin technology (Bayer, 1990; Bashir, 2001; Niemeyer, 1999; Schechter, 1999; Casalini, 1997).
Monomeric avidin may be obtained by immobilizing tetrameric avidin molecules on to a matrix followed by denaturation and renaturation (Henrikson, 1979; Kohanski, 1990). The process for denaturation and renaturation is fairly expensive with loss of non-immobilized avidin subunits and the consumption of a large quantity of expensive reagents in the denaturation step. Furthermore, the high stability of avidin and the strong interfacial interactions between avidin subunits make complete denaturation challenging. Any remaining tetrameric avidin molecules in the matrix can lead to reduced recovery of biotinylated molecules during elution (Kohanski, 1990).
Although streptavidin and avidin have similar three-dimensional structures and biotin binding properties, development of monomeric streptavidin is considered more challenging for at least two reasons. First, streptavidin has stronger subunit interfacial interactions than avidin (Bayer, 1996, Waner, 2004). It is more difficult to weaken this strong interface interaction. Second, monomerization of streptavidin may result in the surface exposure of hydrophobic residues, which normally would be buried at the interface in tetrameric streptavidin. This can potentially affect the solubility of the monomeric streptavidin and lead to re-association of the monomers. Solubility is less of an issue for avidin, which is a glycosylated protein with a carbohydrate chain in each of the avidin subunits.
However, engineered versions of monomeric avidin and streptavidin (Qureshi, 2001; Qureshi, 2002) have been developed. These mutations led to weak H-bonding interactions between streptavidin and biotin, and also affected subunit interactions at the interface. However, the low affinity of this mutein towards biotin (Kd=1.7×10−6M) makes it less than ideal.
Streptavidin is a homo-tetrameric molecule with a biotin binding site in each subunit (Green, 1990). The three dimensional structure of streptavidin (Hendrickson, 1989; Weber, 1989) suggests that a complete biotin binding pocket in each subunit requires the contribution of a tryptophan residue (Trp-120 for streptavidin and Trp-110 for avidin) from an adjacent subunit. Site-directed mutagenesis studies also demonstrate the importance of this residue for tight biotin binding and subunit communications (Freitag, 1998; Sano, 1995; Laitinen, 2001). In the case of avidin, the first generation of engineered monomeric avidin can exist in the monomeric state only in the absence of biotin (Laitinen, 2001). This problem has been solved by the development of a monomeric avidin (Laitinen, 2003), which carries two mutations (N54A, W110K). Structural alignment of avidin and streptavidin indicates that these two residues correspond to D61 and W120 in streptavidin (Livnah, 2003). However, a streptavidin mutein (AK mutein) which carries the corresponding double mutations (D61A, W120K) does not become monomeric (Wu, 2005). Although AK mutein shows reversible biotin binding property and monomeric behavior on the SDS-polyacrylamide gel, it clearly exists in the oligomeric state in solution.
The challenges in producing a monomeric streptavidin mutein, which has useful biotin binding properties, have not yet been completely addressed. There is a need in the art for a streptavidin mutein that may be produced in a soluble and functional form and that reversibly binds to biotinylated biomolecules, which can then be used for capture and/or purification of biotinylated biomolecules.